Process



Patented Feb. 10, 1948 BROCESSFOR PREPARING PHOS- PHORILATED THIAMIN Ben Maizelandrfiving Ger-son; Chiagm? 1112, gas signers "toWico Products Company, Chicago, Ill.

No Drawing, Application July 7,1945; Serial No. 603,150-

8 Claims. (omen-251) Our invention rei'ates to the production of r which the hydroch'l oric acidis not present 01' The synthesisof co-carboxylas'e' or co-carboxylass concentrates has heretofore been known, one well "known method" comprising, in general} reacting avitam'in B1 orthiaminsalt; such as thi- -aniin hydrochloride, with a mixture of orthophosphoric acid and sodium" pyrophosphate at a temperature between about 100 and 200 dgrees 6.} followed by certain recovery and purification procedures; quantities 'of' thiam'ln' salts-; for'-'example; 025 gram, reasonably satisfactory results are obtained and the" reaction can be carried out without undue di flicul-ties although said linown processhas other serious disadvantages among may beqnentionecl the dihiculty of separatingthe desired coc'arb'oxylase from the solubl pliosphates in the reaction mixture; a procedure involving multiple crystallizations from solvents' whiclr "is" not only cum-bersomebut causes' lossiri activity of the cccarboxylase.

' The preparation of co cai boxylase and' similar products in gdod 'yl'elds -and" -by satisfactory procedures presents seriouszvproblemsp In the'first place; the water content of the reaction mixture must be reduced to annmimum since the co carboxylase is l'iydrclyzed bywater. Inthe sec pl'ace, solutions containin'g high concentratviscous and thesatisfactory' incorporation (IF-admixture therewith of thethiamin salt is diflicult even at:elevated temperatures. Due; in part' at least, to the high viscosity oftherreaction mixture; the latter foams veaztiremely a result of therliberatroniofhydrochloric acid from 'the thlaminihydrochloride:ciurine thereactions when attempts are maue to: ractice" the; reviously described: "known 2* process using; however; maceriaIIWgreater quantitiesof thiamin' hydrochloride inthe reaction; fori'example oi -the order-of 25: grams, more-or less, the vfc iamirig:i roblentbecomes1 almost": insurmountable; The enormous quantities of: foam-1 which". are Iiberateu i around each particle-of thlamini -makc itiexceedinglwdiffi'cultato break? up individual lumps of" thiamin without very vigorous stirring, and the latter is When working with very small natureof" the reaction mixture. Still further problems are met= inzconnection'with the neutralization'of the reaction mixture, both thiamin and 'co-carboxylasebeing sensitive to alkalies or alkaline materials. Finally, due to the fact that co-carboxylase is' very' soluble, difliculties are en'- countered in separating the same from undesired solubles present in the-reaction mixture.

In accordance with our present invention, phosphorilated esters of "thiamin may be prepared in sucha manner that not only arethe problems previously enumerated obviated at least in large measure in'the' step ofcarryin'g out the phosphorilation of'the thlamin but," in addition; the separation and recovery of the phosphorilated thiamin are-markedly facilitated.

In general; inaccordance'with our invention,

co-carbOxyIase' is: formed by reacting a thiamin salt, such as thephosphateor sulphate but more particularly a hydrohalilie' su'chas the'hydrobromide or the' hydrochloride; especially the latter, at elevated temperatures, ofv-the order of 100-200 degrees C., and particularly at 120-140 degrees C.', with ph'osph'oricacidcontaining 75%to 81% 'oflP'zO'sand, particularly, substantially 80% of P205. The reactlon proceeds smoothly and easily and produces excellnt'yields of co-carboxylase, namely; of the order o 90-95%'; based upon the amount; of thfamin hydrochloride or other thia- 'min sailtr The phosphoric acid con'talning 75 to 81% of P205; and particularly substantlally30 %"of P205, may be prepared in any" convenient manner as, for example, by dissolving the requisite amount ofPzOs in regular commercial se-called 75% orthophosphorlc acid whichlattcr contains approx imately 50% of POsa- In certain cases, it lsadvantageous to dissolve thethiam-in hydrochloride or-other thl'amin salt in commercial 75% orthophosphoric acid, a phosphoric acidcontaining about "80%' of PsOs being added'in sufficlent amount' to produce' the requisite content ofPzOt,

' the waterin the '75% orthophosphoric acid "he'- very hard to accompllsn sbecauseof' the' viscous in'g changed to orthophosphoric acid; In large scale operations, this" procedure is preferred since it permits-bettercontrol of the reaction;

When the phosphorilating reaction is complete, which is usually-in about iew minutes to about 2 hours, depending-upon the size of the batch,

therea'ction mixture-ls allowed to cool to about 30-40 degrees-0., and then'is poured into a'mix ture of ice and water at/such a-rate that the-tmperat'ure preferably does not 'ris'eappreciably above about 50 "degrees By -so proceeding, hydrolysis is substantially" avoided and the final product comprises co-carboxylase. On the other hand, if cooling is omitted, the temperature, on the addition of water, may rise as high as about 140 degrees C. and the final product comprises the monophosphoric acid ester Of thiamin as distinguished from the co-carboxylase. It will be seen, therefore, that, by this very simple procedure, the process can be carried out to produce either of the aforesaid phosphorilated thiamin pro-ducts, depending upon which is desired. The recovery and purification steps may then be effected in Various ways, as, by way of illustration, described in the following illustrative examples.

In order to achieve the full advantages of the invention, for each mol of thiamin hydrochloride or other salt used in the reaction, there should be employed at least, and preferably in some excess thereof, that amount of the phosphoric acid containing substantially 80% of P205 which is necessary to esterify the thiamin to produce the co-carboxylase, to take up the mol of water which is formed during the reaction and to form orthophosphoric acid therewith, to react with any water of hydration which may be present in the thiamin salt, and to replace the hydrochloric acid in case thiamin hydrochloride is utilized as .one of the reactants. Good results are obtained by using of the order of equal weights of thiamin hydrochloride and 80% of P205 phosphoric acid but it is preferred to employ at least twice as much and particularly five to seven times as much 80% of P205 phosphoric acid than thiamin hydrochloride, by weight.

The following examples are illustrative of the process of the present invention. It will be understood that, within the spirit of the teachings and guiding principles disclosed herein, certain changes may be made without departing from the scope of the invention as set out in the claims.

Example 1 C. and is slowly poured in 500 grams of crushed ice containing justenough water to permit agitation. The reaction mixture contains the desired co-carboxylase, orthophosphoi'ic acid, and water. During the reaction, almost all of the hydrochloric acid present in thethiamin hydrochloride is driven off as a gas. Any residual hydrochloric acid may be removed by subjecting the reaction mixture to a vacuum, preferably immediately after the completion of the reaction. To remove the orthophosphoric acid, a slurry of calcium car- ,bonate is added in small successive amounts until .a pH of about 4 to about 5.5 is reached, The mass is then filtered, washed, if desired, with about 200 cc. of hot water to remove adhering liquid, and the filtrate and washings are evaporated, preferably in vacuo, to a volume of about 60 cc. Any additional precipitate which forms is removed by filtration. To the filtrate there is and ethyl alcohol in the'ratio of two parts of the ether to one part of the alcohol, by volume. The

solution is cooled to about 5 degrees C. wherethen added 250 cc. of a mixture of ethyl ether action mixture may be treated as described in part (a) hereof to recover the monophosphoric acid ester of thiamin and the latter may be converted into its'hydrochloric acid salt in the manner described in part (b) hereof.

Example 2 (a) 35 grams of vthiamin hydrochloride are placed in a one-liter round-bottom flask, equipped with an agitator, 20 grams of 75% orthophosphoric acid are added, and the mixture is heated, with stirring, at about 100 degrees C. until the thiamin hydrochloride is dissolved. Thereupon, 420 grams of phosphoric acid containing P205, which has previously been heated to -140 degrees C., are slowly added, with stirring, over a period of about 15 minutes. The reaction mixture is then allowed to cool to about 30-40 degrees C. and is then treated in the manner described in Example 1, including partsib) and (c).

As indicated in the aboveexamples, the reaction product is treated with a slurry of calcium carbonate. Powdered calcium carbonate maybe employed but it is preferred to use an aqueous slurry in order to avoid the occurrence of lumping. Since the reaction which occurs upon the addition of the calcium carbonate is relatively slow and evolves little heat, it is usually not necessary to cool the reaction mixture during the addition of the calcium carbonate. In place of calcium carbonate, compounds of other alkaline earth metals which form insoluble phosphates may be utilized. Furthermore, hydrated lime may be employed but, in such case, it is generally advisable to agitate and cool the reaction mixture during the addition of the hydrated lime.

After the addition of the calcium carbonate slurry, instead of proceeding in the manner described in the foregoing examples, the entire reaction mixture may, if desired, be dried, as, for example, in a vacuum shelf drier. Where the presence of calcium phosphate is not objectionable, the resulting dried product may be used as such, or the calcium phosphate may be separated adding a member selected from the group consisting of hydrated lime and calcium carbonate until the reaction mixture has a pH of about '4 to about 5.5, filtering, evaporating the filtrate to a low volume, removing any precipitate which forms, adding a mixture of ethyl alcohol and ethyl ether, and recovering the phosphorilated thiamin in the form of crystals.

2. The process which comprises reacting a.

thiamin salt with an excess of phosphoric acid containing substantially 80% of P205 at a temperature of about 120 to about 140 degrees C., cooling the reaction mixture to about -40 derees C., adding the same to crushed ice, and adding an aqueous slurry of calcium carbonate until the reaction mixture has a pH of about 4 to about 5.5.

3. The process which comprises reacting a thiamin salt with an excess of phosphoric acid containing substantially 80% of P205 at a temperature of about 120 to about 140 degrees C., cooling the reaction mixture to about 30-40 degrees C., adding the same to crushed ice, removing any residual hydrochloric acid under vacuum, adding an aqueous slurry of calcium carbonate until the reaction mixture has a pH of about 4 to about 5.5, filtering, evaporating the filtrate to a low volume, removing any precipitate which forms, adding a mixture of ethyl alcohol and ethyl ether, and recovering the phosphorilated thiamin in the form of crystals.

4. The process which comprises dissolving a thiamin salt in an orthophosphoric acid containing approximately 50% of P205, adding a phosphoric acid product thereto containing not substantially less than 80% of P205 in amount sufficient to convert the water present in said firstmentioned orthophosphoric acid to ortho-. phosphoric acid, and reacting the resulting mixture at a temperature within the range of about 100 to about 200 degrees C.

5. The process which comprises dissolving a thiamin salt in an orthophosphoric acid containing approximately 50% of P205, adding a phosphoric acid thereto containing approximately 80% of P205 in amount sufiicient to convert the water present in said first-mentioned orthophosphoric acid to orthophosphoric acid, reacting the resulting mixture at about 120 to about 140 degrees C., cooling the reaction mixture to about 30-40 degrees C., adding the same to crushed ice, and adding an aqueous slurry of calcium carbonate until the reaction mixture has a pH of about 4 to about 5.5.

6. The process which comprises dissolving a thiamin salt in an orthophosphoric acid containing approximately 50% of P205, adding a phosphoric acid thereto containing approximately 80% of P205 in amount sufiicient to convert the water present in said first-mentioned orthophosphoric acid to orthophosphoric acid, reacting the resulting mixture at about 100 to about 200 degrees C., cooling the reaction mixture to about 30-40 degrees C., adding the same to crushed ice, adding a compound of an alkaline earth metal which forms an insoluble phosphate until the reaction mixture has a pH of about 4 to about 5.5.

7. The process which comprises dissolving a thiamin salt in an orthophcsphoric acid containing approximately 50% of P205, adding a phosphoric acid thereto containing approximately of P205 in amount suificient to convert the water present in said first-mentioned orthophosphoric acid to orthophosphoric acid, reacting the resulting mixture at about to about 200 degrees C., cooling the reaction mixture to about 30-40 degrees C., adding the same to crushed ice, removing any residual hydrochloric acid under vacuum, adding an aqueous slurry of calcium carbonate until the reaction mixture has a pH of about 4 to about 5.5, filtering, evaporating the filtrate to a low volume, removing any precipitate which forms, adding a mixture of ethyl alcohol and ethyl ether, and recovering the phosphorilated thiamin in the form of crystals.

8. The process which comprises dissolving thiamin hydrochloride in an orthophosphorie acid containing approximately 50% of P205, adding a phosphoric acid thereto containing approximately 80% of P2O5-il'l amount suflicient to convert the water present in said first-mentioned orthophosphoric acid to orthophosphoric acid, reacting the resulting mixture at about to about degrees C., cooling the reaction mixture to about 30-40 degrees C., adding the same to crushed ice, removing any residual hydrochloric acid under vacuum, adding an aqueous slurry of calcium carbonate until the reaction mixture has a pH of about 4 to about 5.5, filtering, evaporating the filtrate to a low volume, removing any precipitate which forms, adding a mixture of ethyl alcohol and ethyl ether, and recovering the phosphorilated thiamin in the form of crystals.

BEN MAIZEL. IRVING GERSON.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Name Date Tauber Jan. 23, 1940 Jour. of the Amer. Chem. Soc., vol. 60 (1938), pages 2263-2264.

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